WO2004058817A1 - Nouvelles proteines et leur utilisation - Google Patents

Nouvelles proteines et leur utilisation Download PDF

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Publication number
WO2004058817A1
WO2004058817A1 PCT/JP2003/016655 JP0316655W WO2004058817A1 WO 2004058817 A1 WO2004058817 A1 WO 2004058817A1 JP 0316655 W JP0316655 W JP 0316655W WO 2004058817 A1 WO2004058817 A1 WO 2004058817A1
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WIPO (PCT)
Prior art keywords
protein
seq
amino acid
cancer
acid sequence
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PCT/JP2003/016655
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English (en)
Japanese (ja)
Inventor
Eiji Sunahara
Takafumi Ishii
Koji Yamamoto
Shuji Sato
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Takeda Pharmaceutical Company Limited
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Application filed by Takeda Pharmaceutical Company Limited filed Critical Takeda Pharmaceutical Company Limited
Priority to CA002511522A priority Critical patent/CA2511522A1/fr
Priority to US10/540,394 priority patent/US20060110393A1/en
Priority to AU2003292774A priority patent/AU2003292774A1/en
Priority to EP03768194A priority patent/EP1577322A4/fr
Publication of WO2004058817A1 publication Critical patent/WO2004058817A1/fr
Priority to US12/559,938 priority patent/US20100008926A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a novel protein, a polynucleotide encoding the same, a method for producing the same, a cancer preventive / therapeutic agent or diagnostic agent, an apoptosis promoting agent, a cancer preventive / therapeutic agent, or screening for an apoptosis promoting agent.
  • a novel protein a polynucleotide encoding the same, a method for producing the same, a cancer preventive / therapeutic agent or diagnostic agent, an apoptosis promoting agent, a cancer preventive / therapeutic agent, or screening for an apoptosis promoting agent.
  • Recent advances in microarray and oligonucleotide array technologies have enabled comprehensive analysis of gene expression. It has also been predicted that the disease state of cancer can be evaluated by gene microarray profiling, and in fact, it has been reported that leukemia can be classified by gene expression profile in leukemia. To clarify gene expression profiles of individual cancer tissues and accumulate their classification to predict responsiveness to specific cancer treatments and to discover new drug discovery target proteins for specific cancers It will be possible. Specifically, when the expression of a certain protein is increased in a certain cancer, the expression level of the protein is decreased in a newly diagnosed antigen-positive patient; Antitumor activity can be induced by methods such as suppressing functions, and (iii) eliciting a host immune response to the protein.
  • Semaphor in family is a large family of proteins, both secreted and membrane-bound, with at least 19 vertebrate and three invertebrate genes reported (Cell 97). Vol. 551-552, 1999).
  • Semaphor in family is a widespread typified by neural axon guidance and synapse formation It is known to be involved in the peripheral neurogenesis process.
  • the involvement of semaphor in family in the immune system (Trends in I. unol. 22, 670-676, 2001) and its involvement in 11 organ development and angiogenesis are becoming apparent.
  • Semaphor in 3B and Semaphor in 3F derived from human belonging to Semaphor in family have been reported as tumor suppressor genes (Proc. Natl Acad. Sc. USA 98, 13954-13959, 2001; Cancer Res. 62, 542-546, 2002, Cancer Res. 62, 2637-2643, 2002).
  • Semaphor in 3C has been reported to be upregulated in human lung cancer tissues (J. Surg. Oncol. 72, 18-23, 1999, Proc. Natl Acad. Sc. USA 94, 14713-718, 1997). Semaphor in 3E has been reported to be expressed in metastatic cells (Cancer Res. 58: 1232-1244, 1998).
  • Semaphorin 4B having 41% homology to Semaphorin4D at the amino acid level (hereinafter sometimes abbreviated as SEMA4B) is registered in GenBank as a gene predicted from the genome sequence (GenBank Accession No. XMJ44533). SEMA4B has been reported as one of the genes whose expression increases under hypoxic conditions (TO 02/46465). In addition, it has been reported that hundreds of nucleotide sequences, including SEMA4B, can be used to diagnose lung cancer or to search for compounds that treat lung cancer based on gene chip analysis (W02 / 86443) SEMA4B and amino acid levels N0V7 with 93% homology has been reported to be expressed in cancer (W002 / 06329).
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, found a novel gene whose expression is significantly increased in lung cancer tissues, and the antisense oligonucleotide of this gene promotes apoptosis of cancer cells. I also found out. Further studies based on this knowledge have led to the completion of the present invention.
  • a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the polynucleotide of (4) or a part thereof,
  • (21) A method for screening a compound or a salt thereof that inhibits the expression of the protein according to (1), which comprises using the protein or partial peptide thereof or a salt thereof according to (1);
  • (22) a kit for screening a compound or a salt thereof that inhibits the expression of the protein of (1), which comprises the protein of (1) or a partial peptide thereof or a salt thereof,
  • (22a) a compound or a salt thereof, which inhibits the expression of the protein of (1), obtained by using the screening method of (21) or the screening kit of (22);
  • (22b) a medicine comprising the compound of (22a) or a salt thereof described above; (23) the protein gene of (1), characterized by using the polynucleotide of (4) above; A method for screening a compound that inhibits the expression of
  • an apoptosis-promoting agent comprising a substance that inhibits the expression of the protein or the partial peptide thereof according to (1) or the expression of the gene of the protein,
  • an apoptosis-promoting agent comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof;
  • a prophylactic / therapeutic agent for cancer comprising an antibody against a protein having the same or colder amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof ,
  • a cancer cell growth inhibitory agent comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof ,
  • (31) a base complementary or substantially complementary to the base sequence of a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1
  • (32) a medicament comprising the polynucleotide according to (31); (33) a medicament according to (32), which is an apoptosis-promoting agent;
  • an apoptosis-promoting agent which comprises a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding a partial peptide thereof; Screening kit,
  • an amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 A substance that inhibits the expression of a protein containing a sequence or a partial peptide or a salt thereof; (ii) a substance that inhibits the expression of a gene of the protein or a partial peptide thereof; or (iii) a protein or a part thereof.
  • a method for promoting apoptosis of cancer cells which comprises administering an effective amount of a peptide or a salt thereof to the subject.
  • SEQ ID NO: 1 SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10
  • a method for promoting apoptosis of cancer cells which comprises inhibiting the expression of a salt thereof, or inhibiting the expression of the gene for the protein or its partial peptide.
  • a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 (hereinafter referred to as the present invention) Protein or the protein used in the present invention) is a cell of a human warm-blooded animal (eg, guinea pig, rat, mouse, chick, egret, bush, sheep, hidge, monkey, etc.).
  • a human warm-blooded animal eg, guinea pig, rat, mouse, chick, egret, bush, sheep, hidge, monkey, etc.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is 95% or more, preferably about 98% or more, preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequences having a homology of 99% or more.
  • an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 1 And a protein having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 examples include an amino acid sequence having 99.9% or more homology with the amino acid sequence represented by SEQ ID NO: 4 .
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 4 is preferred. ' Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7 include an amino acid sequence having 99.9% or more homology with the amino acid sequence represented by SEQ ID NO: 7.
  • Examples of the protein containing the f-amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 include, for example, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 described above. And a protein having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 7 is preferred.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 10 includes, for example, an amino acid sequence having 99.9% or more homology with the amino acid sequence represented by SEQ ID NO: 10 Is mentioned.
  • proteins having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 10 include, for example, those substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 10
  • a protein having an amino acid sequence and having substantially the same activity as a protein having an amino acid sequence represented by SEQ ID NO: 10 is preferred.
  • the homology of amino acid sequences can be calculated using the homology calculation algorithm NCBI BLAST (National-Center for Biotechnology Information Basic Basic Alignment Search).
  • Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, the activity of the protein of the present invention may be equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Although preferred, the quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • Examples of the protein used in the present invention include: (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (eg, about 1 to 50, preferably 1 to 50) An amino acid sequence in which about 30 amino acids have been deleted, more preferably about 1 to 10 amino acids, and still more preferably about 1 to 5 amino acids, (ii) SEQ ID NO: 1 1 or 2 or more (for example, about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, more preferably about 1 to 50, ) Amino acid sequence; (iii) one or more (for example, about 1 to 50, preferably about 1 to 30 and more preferably about 1 to 50 amino acids in the amino acid sequence represented by SEQ ID NO: 1) Is an amino acid sequence having about 1 to 10 amino acids, more preferably (1 to 5) amino acids, and (iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, An amino acid sequence in which about 1 to 50, preferably about 1 to 30, more
  • (2) an amino acid sequence in which one or two amino acids in the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 are deleted; SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 has 1 or more amino acid sequences (for example, about 1 to 50, preferably about 1 to 30, more preferably An amino acid sequence to which about 1 to 10 amino acids are added, and more preferably (1 to 5) amino acids;
  • (iii) SEQ ID NO: 4, SEQ ID NO: 0.7 or SEQ ID NO: 10 (Iv) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10; An amino acid sequence in which one amino acid has been replaced with another amino acid, or (V) an amino acid that combines them So-called mucins such as proteins containing sequences are also included.
  • mucins such as proteins containing sequences are also included
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary terminus) according to the convention of peptide notation.
  • the protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (-C00H), carboxylate (-C00-), amide (-C0NH) 2 ) or ester (-C00R).
  • R in the ester is, for example, methyl, ethyl, n-propyl Le, isopropyl, C, such as n- butyl, _ 6 alkyl groups, for example, cyclopentyl Le, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, shed - C 6, such as naphthyl - 12 Ariru group , for example, benzyl, phenyl, such as phenethyl - _ 14 Ararukiru groups such as C Bok 2 a one Nafuchiru al kill such as an alkyl group or a flying one naphthylmethyl, etc.
  • Pipa Roi Ruo carboxymethyl group is used.
  • the protein used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention.
  • the ester in this case for example, the above-mentioned ester at the terminal or the like is used.
  • the protein used in the present invention the amino acid residue (e.g.,. Mechionin residues) of N-terminal Amino group protecting groups (e.g., such as C M Ashiru group such Arukanoiru such formyl group, Asechiru group) N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation,
  • N-terminal Amino group protecting groups e.g., such as C M Ashiru group such Arukanoiru such formyl group, Asechiru group
  • N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation
  • Substituent on the side chain of amino acid in the molecule eg, ⁇ H, —SH, amino, group, imidazole group, indole group, Guanijino group, etc.
  • a suitable protecting group e.g., ho mill group, those protected by Asechiru like Ashiru groups such
  • protein used in the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 4, and a protein containing the amino acid sequence represented by SEQ ID NO: 7.
  • a protein containing the amino acid sequence represented by SEQ ID NO: 10, and the like are examples of the protein used in the present invention.
  • the partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and is preferably any one having the same properties as the protein used in the present invention described above. It may be something.
  • At least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably Peptides having an amino acid sequence of preferably 100 or more, most preferably 200 or more are used.
  • the partial peptide used in the present invention may have one or two or more amino acids in the amino acid sequence thereof (preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5 ) Amino acids are deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5 ) Amino acids, or 1 or 2 or more (preferably about 1-20, more preferably about 1-10, and more preferably about 1-10 amino acids) in the amino acid sequence. 5)) amino acids, or 1 or 2 or more (preferably about 1 to 20; more preferably about 1 to 10; more preferably several) in the amino acid sequence. (More preferably about 1 to 5) amino acids are replaced with other amino acids It may be.
  • the partial peptide used in the present invention may be anything Re at the.
  • the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) in addition to the C-terminal, and the N-terminal amino acid residue, as in the above-mentioned protein used in the present invention.
  • carboxyl group or carboxylate
  • N-terminal amino acid residue as in the above-mentioned protein used in the present invention.
  • methionine residue whose amino group is protected by a protecting group, N-terminal cleavage in vivo, dalminamine residue generated by pyroglutamine oxidation, side chain of amino acid in the molecule
  • glycopeptide such as a so-called glycopeptide.
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal salt
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Acids, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • the protein or its partial peptide or a salt thereof used in the present invention can be produced from the cells or tissues of the above-mentioned human ⁇ warm-blooded animal by a known method for purifying protein. It can also be produced by culturing a transformant containing the encoding DNA. It can also be produced according to the peptide synthesis method described below. '
  • the human or mammalian tissues or cells are homogenized, extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include, for example, chlorotyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4_ (2', 4'dimethoxyphenyle Fm oc aminoethyl) phenoxy resin and the like.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the target protein according to various condensation methods known per se.
  • protein or partial peptide is cut out from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or their peptide Obtain the amide form.
  • the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOBt, HOOBt) or pre-protected as a symmetrical anhydride or HOBt ester or HOOBt ester. It can be added to the resin after the activation of the amino acid.
  • a racemization inhibitor additive eg, HOBt, HOOBt
  • pre-protected as a symmetrical anhydride or HOBt ester or HOOBt ester it can be added to the resin after the activation of the amino acid.
  • the solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, trifluoroethanol, etc.
  • Alcohols, sulphoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane, tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. Used.
  • the reaction temperature is appropriately selected from a range known to be usable for the protein bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isopolnyloxycarbonyl, 4-methoxybenzyloxyl-ponyl, C 1 _Z, Br—Z, and adaman Tiloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group may be, for example, an alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (e.g., benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, etc. Can be protected.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohex
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower (Cw) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, and an ethoxycarbonyl group are used.
  • a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C 1 2 one B zl, 2_ nitrobenzyl, B r- Z, such as t one-butyl is used.
  • protecting group for histidine imidazole for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used. .
  • Examples of the activated carbonyl group of the raw material include, for example, corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t)].
  • alcohol for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t
  • As the activated amino group of the raw material for example, a corresponding phosphoramide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc.
  • Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, treatment with base using diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used.
  • the elimination reaction by the above acid treatment is generally performed at a temperature of about 120 ° C. (to 40 ° C.).
  • a cation scavenger such as anisol, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • a cation scavenger such as anisol, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
  • the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 4
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group which should not be involved in the reaction of the raw material, the protection group, the elimination of the protective group, and the activation of the functional group involved in the reaction can be appropriately selected from known groups or known means.
  • an amide of a protein or partial peptide for example, first, amidating the 0! -Hydroxyl group of the carpoxy terminal amino acid and protecting it, and then attaching a peptide (protein) chain to the amino group side of the desired After extending to the chain length, a protein or partial peptide from which only the N-terminal ⁇ -amino group-protecting group of the peptide chain has been removed and a partial peptide and only the C-terminal lipoxyl group-protecting group have been removed. Are produced, and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • an ester of a protein or peptide for example, after condensing the ⁇ -lipoxyl group of the terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of a protein or peptide, An ester of the desired protein or peptide can be obtained.
  • the partial peptide used in the present invention or a salt thereof can be prepared by a known peptide synthesis method, or by converting the protein used in the present invention into a suitable peptide. It can be produced by cutting with lyase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide which can constitute the partial peptide used in the present invention is obtained by condensing an amino acid and the remaining part, and when the product has a protecting group, removing the protecting group to obtain the desired peptide. Can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (V).
  • the partial peptide used in the present invention can be purified and isolated by combining crystals and the like.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, a known method or Can be converted into a free form or another salt by a method analogous thereto.
  • the polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein used in the present invention.
  • it is DNA.
  • the DNA may be any of genomic DNA, genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Also, the cell described above. Reverse RNA was directly prepared using total RNA or mRNA fraction prepared from tissue. It can also be amplified by the Transcriptase Polymerase Chain Reaction (hereinafter referred to as RT-PCR method).
  • the DNA encoding the protein used in the present invention includes, for example, (i) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a highly stringent condition with the nucleotide sequence represented by SEQ ID NO: 2 A protein having a base sequence that hybridizes under the following, and encoding a protein having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 1;
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 2 and 95% or more, preferably about 98 or more, and more preferably DNA containing a base sequence having about 99% or more homology is used.
  • Examples of DNA that can hybridize with the base sequence represented by SEQ ID NO: 5 under high stringent conditions include, for example, a base having 99.9% or more homology with the base sequence represented by SEQ ID NO: 5 DNA containing a sequence is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 8 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 8 DNA containing a base sequence having 99.9% or more homology is used.
  • Examples of a DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 11 under high stringent conditions include, for example, 99.9% or more homology with the nucleotide sequence represented by SEQ ID NO: 11
  • a DNA containing a base sequence or the like is used.
  • Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringency conditions.
  • High stringency end conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C.
  • a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM
  • a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C.
  • the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 includes a DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO:
  • the DNA containing the nucleotide sequence represented by SEQ ID NO: 5, etc. is used as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4.
  • DNA or DNA containing the nucleotide sequence represented by SEQ ID NO: 6; and (iii) DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 7 includes SEQ ID NO: DNA containing the nucleotide sequence represented by SEQ ID NO: 8 or DNA containing the nucleotide sequence represented by SEQ ID NO: 9 encodes (iv) a protein containing the amino acid sequence represented by SEQ ID NO: 10 SEQ ID NO: 11 DNA or SEQ ID NO containing the base sequence that is: such as DN A is used which contains a nucleotide sequence represented by 12.
  • the polynucleotide (eg, DNA) encoding the partial peptide used in the present invention includes a base sequence encoding the partial peptide used in the present invention described above. Any material containing a row may be used. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a part of the DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11.
  • the DNA of the present invention which comprises a DNA having the sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 and a nucleotide sequence that hybridizes under high stringent conditions
  • DNA containing a part of DNA encoding a protein having substantially the same activity as a protein is used.
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 has the same significance as described above.
  • DNA that completely encodes the protein or partial peptide used in the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of the cloning and expression of the DNA encoding the same).
  • DNA amplified by the PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or a DNA incorporated into an appropriate vector is used for the cloning of the present invention.
  • Selection can be carried out by hybridization with a DNA fragment coding for a part or the entire region of the protein or labeled with synthetic DNA.
  • the hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
  • it can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be converted using PCR, a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method. , Gapped duplex method, Kunkel method, etc. You can do it in the following way:!?
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention includes, for example, (a) cutting out a target DNA fragment from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting to
  • the vector examples include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast ( For example, pSH19, pSH15), bacteriophage such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ⁇ 1_11, ⁇ XT1, pRcZCMV, pRc / RSV, pc DNA IZNeo is used. '
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR promoter when animal cells are used as hosts, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. ,
  • CMV cytomegalovirus
  • SRa promoter cytomegalovirus promoter
  • the host is Escherichia, trp promoter, lac promoter, recA promoter,? Otsupu opening motor -, ⁇ ⁇ ⁇ promoter, Ding?
  • the host is a Bacillus genus, SPOL promoter, SP ⁇ 2 promoter, penP promoter, etc.
  • the host is yeast, PHO5 promoter, PGK Promoters, GAP promoters, ADH promoters and the like are preferred.
  • polyhedrin promoter overnight, P10 promoter evening, and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), and the like, if necessary, may be used as the expression vector.
  • the selection Ma one car, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp r) , neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
  • dh fr gene is used as a selection marker by using dh fr gene-deficient Chinese eight muster cells
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a PhoA 'signal sequence, a 0-A signal sequence, etc.
  • a signal sequence such as an amylase * signal sequence and a subtilisin signal sequence, If the host is yeast, MFo! Signal sequence, SUC2, signal sequence, etc.
  • the host is a animal cell, insulin signal sequence, ⁇ ; —interferin signal sequence, antibody molecule A signal sequence or the like can be used.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, konjac, animal cells and the like are used.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus subtilis MI114 Gene, 24, 255 (1983)
  • 207-21 Journal of Biochemistry, 95, 87 (1984)] and the like are used. .
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD-5D, 20B—12, Schizosaccharomyces poinbe N CYC 1913, NCYC 2036, Pichia pastori (Pichia pastoris) KM 71 or the like is used.
  • insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of night rob moth (Spodoptera frugiperda cell; Sf cell), an MG1 cell derived from the midgut of Trichoplusia ni, and a High Five derived from the egg of Trichoplusia ni TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. If the virus is NPV, a silkworm-derived cell line (Bombyx mori cell; BmN cell) or the like is used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (above, Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
  • insects for example, silkworm larvae are used [Maeda et al., Neichia- (Nature), 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CH ⁇ (hereinafter, CHO (dh fr ”) Cells, mouse L cells, mouse AtT-20, mouse myeoma cells, mouse ATDC 5 cells, rat GH3, human FL cells, and the like.
  • CHO Chinese hamster cell CHO
  • yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be.
  • Insect cells or insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-. 55 (1988).
  • a liquid medium is suitable as a medium for cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • examples of the nitrogen source include ammonium salts, nitrates, corn chip liqueur, peptone, casein, meat extract, soybean meal, and potatoes.
  • examples of inorganic or organic substances and inorganic substances such as an extract include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8. .
  • a culture medium for culturing the genus Escherichia for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be applied.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • Burk Folder (Burkholder) Minimal Medium [Proc. Natl. Acad. Sci. USA, 77.
  • the pH of the medium is preferably adjusted to about 5-8.
  • the cultivation is usually performed at about 20 ° C. to 35 t for about 24 to 72 hours, and aeration and stirring are added as necessary.
  • the culture medium When culturing an insect cell or a transformant in which the host is an insect, the culture medium may be supplemented with Grace's Insect Medium (Nature, 195,788 (1962)) with appropriate addition of immobilized 10% serum or other additives. Are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the culture medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , 8, 396 (1959)], RPMI 1640 medium [The
  • the pH is between about 6 and 8.
  • Culture is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the protein of the present invention can be produced in the cells of the transformant, in the cell membrane, or outside the cells. '
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and then sonicated, lysozyme and Z or freeze-thawed. After the cells or cells are disrupted, a method of obtaining a crude oil effluent of the protein by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM. If protein is secreted into the culture, the cells may be secreted after the culture by a method known per se. Separate the supernatant from the cells or cells and collect the supernatant.
  • the protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, etc.
  • Method using difference in electric charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, reverse phase high performance liquid chromatography, etc.
  • a method utilizing the difference in hydrophobicity a method utilizing the difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the protein thus obtained when it is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or analogous thereto Depending on the method, it can be converted into a free form or other salts.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using a specific antibody.
  • the antibody against the protein or partial peptide or a salt thereof used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. Is also good.
  • an antibody against the protein or partial peptide used in the present invention or a salt thereof uses the protein of the present invention as an antigen, Known antibodies Alternatively, it can be produced according to a method for producing an antiserum.
  • the protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible by administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • warm-blooded animals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, an individual with an antibody titer from a mouse is selected, and spleen or lymph nodes are collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [ Nature , 256, 495 (1975)].
  • the fusion promoter include polyethylene daricol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells include warm-blooded animal myeloma cells such as NS_1, P3U1, SP2 / 0, and AP-1, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells to be used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C, for 1 to 10 minutes.
  • anti-immunoglobulin which is obtained by adding a hybridoma culture supernatant to a solid phase (eg, a microplate) onto which a protein antigen is directly or adsorbed together with a carrier, and then labeling with a radioactive substance, an enzyme, or the like
  • a method of adding a hybridoma culture supernatant to the adsorbed solid phase, adding a protein labeled with a radioactive substance, an enzyme, or the like, and detecting a monoclonal antibody bound to the solid phase can be used.
  • the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. )
  • a serum-free medium for culturing eight hybridomas SF M_101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C.
  • the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only antibody is collected using an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody. You can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only antibody is collected using an active adsorbent such as protein A or protein G, and the bond is dis
  • the polyclonal antibody of the present invention can be obtained by a method known per se or a method analogous thereto. Therefore, it can be manufactured. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance of the protein of Example 1 and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier protein and hapten are determined by cross-linking carrier protein to immunization. Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently produced against the hapten.However, for example, serum albumin, thyroglobulin, hemocyanin, etc. are converted to hapten 1 by weight. On the other hand, a method of coupling at a rate of about 0.1 to 20, preferably about 1 to 5 is used. '
  • various condensing agents can be used for force coupling between the hapten and the carrier protein, but a dartalaldehyde, a carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible or with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production during administration.
  • the administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood, of the warm-blooded animal immunized by the above method.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as in the above-described monoclonal antibody separation and purification.
  • Polynucleotides encoding the protein or partial peptide used in the present invention eg, DNAs (hereinafter, in the description of antisense polynucleotides, these DNAs may be abbreviated as DNAs of the present invention)
  • An antisense polynucleotide having a basic or substantially complementary base sequence or a part thereof includes a complementary or substantially complementary base sequence of a polynucleotide (eg, DNA) of the present invention.
  • Any antisense polynucleotide may be used as long as it contains the nucleotide sequence or a part thereof and has an action capable of suppressing the expression of the DNA, and antisense DNA is preferable.
  • the nucleotide sequence substantially complementary to the DNA of the present invention refers to, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention).
  • Base sequences having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology are exemplified.
  • the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a base sequence near the start codon, etc.
  • an antisense polynucleotide which directs RNA degradation by RNaseH it is about 70% or more, preferably about 80% or more, more preferably about 80% or more, complementary to the entire nucleotide sequence of the DNA of the present invention including introns.
  • Antisense polynucleotides having about 90% or more, most preferably about 95% or more homology are each suitable.
  • nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 12 An antisense polynucleotide having a base sequence complementary to or substantially complementary to a base sequence of a DNA containing, or a part thereof, preferably, for example, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: : 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 or a nucleotide sequence complementary to the nucleotide sequence containing the nucleotide sequence containing the nucleotide sequence represented by SEQ ID NO: 12, or a part thereof And an antisense polynucleotide having the same.
  • the antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA should be chemically modified, for example, with phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted with a decorated phosphate residue.
  • the sugar (deoxy report) of each nucleotide may be substituted with a chemically modified sugar structure such as 2, -O-methylation, or the base (pyrimidine, purine) may be chemically modified. And any one that hybridizes to DNA having the base sequence represented by SEQ ID NO: 2.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • an antisense polynucleotide (nucleic acid) corresponding to the gene of the protein of the present invention, which can inhibit the replication or expression of the gene, is cloned or the determined protein is copied. It can be designed and synthesized based on the base sequence information of DNA.
  • Such antisense polynucleotides can hybridize to the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-associated RNA of the present invention.
  • the expression and expression of the protein gene of the present invention can be regulated and controlled.
  • Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, and polynucleotides that can specifically hybridize with the protein-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of protein genes, and is also useful for treating or diagnosing diseases.
  • the term "corresponding" means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene.
  • nucleotide, nucleotide sequence or nucleic acid and a protein usually refers to the amino acids of the protein (as specified by the instructions) derived from the nucleotide (nucleic acid) sequence or its complement.
  • Antisense polynucleotides are polynucleotides containing 2-doxy-D-reports, polynucleotides containing D-reports, or other types of polynucleotides that are N-glycosides of purine or pyrimidine bases.
  • polymers having a non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds provided that the polymer is not contained in DNA or RNA.
  • They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrid, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides).
  • nucleic acid may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such a modification
  • the product may contain methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. May be converted to
  • the antisense polynucleotide of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides that are resistant to degradation of oligonucleoside amides.
  • the antisense polynucleotide of the present invention can be designed, for example, as follows. I.e., to make the antisense polynucleotide more stable in the cell, to enhance the cell permeability of the antisense polynucleotide, to increase the affinity for the target sense strand, and to increase the toxicity.
  • the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Appli cations, CRC Press, 1993.
  • the antisense polynucleotides of the present invention may contain altered or modified sugars, bases, or bonds, are provided in special forms such as liposomes, microspheres, or are applied by gene therapy. Or can be given in additional form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which increase the interaction with cell membranes or increase the uptake of nucleic acids ( Hydrophobic substances such as, for example, phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.).
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. .
  • the protein or partial peptide of the present invention or a salt thereof may be abbreviated as the protein of the present invention
  • a polynucleotide encoding the protein or partial peptide of the present invention eg, DNA
  • DNA a polynucleotide encoding the protein or partial peptide of the present invention
  • the DNA of the present invention may be abbreviated
  • an antibody against the protein or partial peptide of the present invention or a salt thereof hereinafter may be abbreviated as the antibody of the present invention
  • the antisense of the DNA of the present invention The use of the polynucleotide (hereinafter, sometimes abbreviated as the antisense polynucleotide of the present invention) will be described.
  • Pharmaceuticals containing salts or antibodies against the protein of the present invention include, for example, cancers (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, It can be used as a preventive and therapeutic agent for bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, blood tumor, etc., and an apoptosis promoter
  • the expression of the protein of the present invention is enhanced in cancer tissues. Furthermore, when the activity of the protein of the present invention is inhibited, cancer cells undergo apoptosis. Therefore, compounds or salts thereof that inhibit the activity of the protein of the present invention include, for example, cancer (eg, colon) Cancer, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain, blood It can be used as a preventive and therapeutic agent, an apoptosis promoting agent, etc. Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the activity of the protein of the present invention.
  • cancer eg, colon
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the activity of the protein of the present invention.
  • the present invention provides a method for screening a compound or a salt thereof that inhibits the activity of the protein of the present invention, which comprises using the protein of the present invention.
  • a host transformed with a vector containing the DNA encoding the protein of the present invention described above is used.
  • a host for example, animal cells such as COS 7 cells, CHO cells, and HEK293 cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the method of culturing cells capable of expressing the protein of the present invention is the same as the method of culturing the transformant of the present invention described above.
  • -Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • the activity of the protein of the present invention in the case of the above ⁇ (ii) is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more compared with the case of the above (i).
  • the test compound to be tested can be selected as a compound that inhibits the activity of the protein of the present invention.
  • the compound having the activity of inhibiting the activity of the protein of the present invention is useful as a safe and low-toxicity drug for suppressing the physiological activity of the protein of the present invention.
  • compounds that inhibit the expression of the gene of the protein of the present invention or salts thereof include, for example, cancer (eg, colon cancer, breast cancer, lung cancer) , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain cancer, blood tumor, etc.) ⁇ Can be used as a therapeutic agent, apoptosis promoting agent, etc.
  • the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the expression of the protein gene of the present invention.
  • the screening methods include (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein used in the present invention in the presence of a test compound.
  • a screening method characterized by performing a comparison with the case where the method is performed.
  • the expression level of the gene in the cases (ii) and (iv) (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.
  • test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
  • the amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis or ELISA method or a method analogous thereto. It can be measured according to
  • the amount of mRNA can be measured by a known method, for example, by using a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 or a part thereof as a probe.
  • the gene expression in the case (iv) is compared with the case (iii).
  • a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more can be selected as a compound that inhibits the expression of the gene of the protein of the present invention.
  • the screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.
  • Compounds or salts thereof obtained by using the screening method or screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plants.
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention can be used.
  • the compound or its salt that inhibits the activity of the protein of the present invention and the compound or its salt that inhibits the expression of the gene of the protein of the present invention can be, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer) , Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor, blood tumor, etc.) It is useful as a low toxic and safe drug.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer
  • Gastric cancer eg, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor, blood tumor, etc.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals.
  • carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate. A shim or the like is used.
  • compositions for parenteral administration include injections, suppositories, and the like.
  • Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, Includes dosage forms such as intra-articular injections.
  • Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the compound or a salt thereof in a sterile aqueous or oily liquid usually used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents such as alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add of hydrogenated catalyst)] and the like.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add of hydrogenated catalyst)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • Suppositories to be used for rectal administration are prepared by mixing the above-mentioned compound or a salt thereof with a usual suppository base.
  • the above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to conform to the dose of the active ingredient.
  • dosage unit forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg per dosage unit form, especially for injections.
  • the compound contains 5 to 10 O mg, and other dosage forms contain 10 to 25 O mg of the above compound.
  • compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg, mice, rats, puppies, higgs, bushus, puppies, pumas, birds, cats, dogs). , Monkeys, chimpanzees, etc.) orally or parenterally.
  • warm-blooded animals eg, mice, rats, puppies, higgs, bushus, puppies, pumas, birds, cats, dogs.
  • Monkeys, chimpanzees, etc. orally or parenterally.
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.
  • the protein of the present invention may be used.
  • a compound or its salt that inhibits the expression of a quality gene is orally administered, generally, in an adult (with a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1 to 100 mg of the compound or its salt per day is administered. 0-50 mg, more preferably about 1.0-20 mg.
  • the single dose of the compound or a salt thereof varies depending on the administration subject, the target disease, and the like.
  • the gene of the protein gene of the present invention may be used.
  • a compound that inhibits expression or a salt thereof usually in the form of an injection
  • the compound or a salt thereof is injected into the cancer lesion at a rate of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is convenient to administer by In the case of other animals, the dose can be administered in terms of 60 kg of body weight.
  • the antibody of the present invention can specifically recognize the protein of the present invention, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay.
  • the present invention provides a method for quantifying the protein of the present invention in a test solution, which is characterized in that:
  • one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • a monoclonal antibody against the protein of the present invention hereinafter sometimes referred to as the monoclonal antibody of the present invention
  • tissue staining or the like May use the antibody molecule itself, or F (ab ') 2 , Fab, or the Fab fraction of the antibody molecule.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution is measured. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competition method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope e.g., [125 1], [131 1] ', [3 ⁇ 4], C 14 C] and the like.
  • the above enzyme a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase, 3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. .
  • the fluorescent substance examples include cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)), fluorescamine, fluorescein isothiocyanate, and the like. You.
  • cyanine fluorescent dyes eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)
  • fluorescamine fluorescein isothiocyanate
  • fluorescein isothiocyanate examples of the fluorescent substance.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined.
  • Primary reaction and The secondary reaction may be performed in the reverse order, simultaneously, or at a different time.
  • the labeling agent and the method of insolubilization can be the same as those described above.
  • the antibody used for the solid phase or the antibody for labeling is not necessarily required to be one type, and two or more types may be used for the purpose of improving measurement sensitivity and the like. A mixture of bodies may be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction differs in the binding site of the protein of the present invention.
  • Antibodies are preferably used. That is, when the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry method. '
  • the competitive method after the antigen in the test wave and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( BZF separation), B, or F labeling is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • BZF separation is performed using polyethylene glycol
  • a solid phase antibody is used as the first antibody
  • An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.
  • the antigen in the test wave and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test wave is Is allowed to react with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of the label in either phase is measured to determine the amount of the antigen in the test wave.
  • the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured.
  • Lithium or the like is preferably used.
  • the system for measuring the protein of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews and documents.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, cancer (eg, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, visceral cancer, brain tumor, blood tumor, etc.) or in the future It can be diagnosed as having a high possibility of being affected.
  • cancer eg, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, visceral cancer, brain tumor, blood tumor, etc.
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • a subject such as a body fluid or a tissue.
  • the preparation of the antibody column used for purifying the protein of the present invention and the protein of the present invention in each fraction at the time of purification were performed. It can be used for detecting proteins, analyzing the behavior of the protein of the present invention in test cells, and the like.
  • the DNA of the present invention can be used, for example, by using it as a probe to produce human or blood-purified animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs). , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in DNA or mRNA that encode the protein of the present invention or a partial peptide thereof.
  • the decreased expression or increased DNA or mRNA is useful as a diagnostic agent for gene overexpression and the like.
  • the above-described genetic diagnosis using the DNA of the present invention can be carried out, for example, by the known Northern hybridization ⁇ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989, Proceedings of the National Academy of Sciences). of the United States of America, Vol. 86, pp. 2766-2770 (1989)).
  • cancer eg, colorectal cancer, breast cancer, lung cancer, prostate
  • Cancer esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain, blood, etc.
  • testis thyroid, kidney, brain, blood, etc.
  • the antisense polynucleotide of the present invention which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and has the function of the protein of the present invention or the DNA of the present invention in vivo. It can suppress the action and induce apoptosis of cancer cells, and can be used for cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, Renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor sputum, blood tumor, etc.) It can also be used as an apoptosis accelerator.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, Renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer,
  • said antisense polynucleotide said prophylactic or therapeutic agents when used in such accelerators, formulated according to a method known per se, also f can be administered, for example, alone or retro the antisense polynucleotides
  • a suitable vector such as a viral vector, an adenovirus vector, or an adenovirus associated virus vector
  • the cells After insertion into a suitable vector such as a viral vector, an adenovirus vector, or an adenovirus associated virus vector, the cells are inserted into a human or mammal (eg, a rat, a egret, a sheep, a pig, a pig, a cat) in a conventional manner. , Dogs, monkeys, etc.) can be administered orally or parenterally.
  • the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.
  • a physiologically acceptable carrier such as an auxiliary agent for promoting uptake
  • the antisense polynucleotide is formulated alone or in combination with a carrier such as ribosome (injection), and is intravenously, subcutaneously, etc. May be administered.
  • the dose of the antisense polynucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.
  • the antisense polynucleotide of the present invention is administered for the purpose of treating breast cancer, it is generally used.
  • an adult body weight 60 kg
  • about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
  • the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • RNA containing a part of the RNA encoding the protein of the present invention a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, etc.
  • the expression of the gene of the present invention can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention in vivo can be suppressed, for example, cancer (eg, colon cancer, breast cancer) , Lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor, blood tumor, etc.) It can be used as a preventive / therapeutic agent, apoptosis promoter, etc.
  • cancer eg, colon cancer, breast cancer
  • prostate cancer esophageal cancer
  • stomach cancer liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer
  • testis cancer thyroid cancer
  • kidney cancer brain tumor, blood tumor, etc.
  • the double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, vol. 41i, p. 494, 2001).
  • the lipozyme can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention.
  • a part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.
  • RNA or lipozyme When the above-described double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. (5) a drug containing the antibody of the present invention
  • the antibody of the present invention has apoptosis-inducing activity of cancer cells, it can be used, for example, in cancers (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, Prevention and treatment of bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, genital cancer, brain tumor, blood tumor, etc. (eg, vaccines, etc.) (preferably lung cancer, ovarian cancer, pentine cancer, etc.) Can be used as an apoptosis promoter, etc.
  • cancers eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, Prevention and treatment of bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, genital cancer, brain tumor, blood tumor, etc. (eg, vaccines
  • the prophylactic / therapeutic agents and promoters for the above-mentioned diseases containing the antibody of the present invention have low toxicity, and can be used as they are as liquids or as pharmaceutical compositions in appropriate dosage forms, in humans or mammals (eg, rats, egrets, higgies). Can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.) to mice, monkeys, cats, cats, dogs, monkeys, etc.). Preferably, it can be administered as a vaccine according to a standard method.
  • the antibody of the present invention may be administered per se or as a suitable pharmaceutical composition.
  • Pharmaceutical compositions used for administration include the antibody of the present invention and salts thereof, and a pharmacologically acceptable carrier, diluent or excipient. It may be. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • compositions for parenteral administration for example, injections, suppositories, vaccines, etc. are used.
  • Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Dosage forms may be included.
  • Such an injection can be prepared according to a known method. Injection preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid commonly used for injections.
  • aqueous liquid for injection for example, physiological saline ice, isotonic solution containing glucose and other adjuvants is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, , Polypropylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduct of hydrogenated cast oil)), etc. Good.
  • a suitable solubilizing agent for example, alcohol (eg, ethanol), polyalcohol (eg, Polypropylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduct of hydrogenated cast oil)), etc. Good.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like.
  • Such a composition is produced by a known method and may contain carriers, diluents or excipients usually used in the field of formulation.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form so as to be compatible with the dosage of the active ingredient.
  • dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody content is usually 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injection, and 10 to 250 mg for other dosage forms. Is preferred.
  • the dosage of the above-mentioned prophylactic / therapeutic agent or modulator containing the antibody of the present invention varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, it is used for the treatment and prevention of adult breast cancer.
  • the amount of the antibody of the present invention per dose is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably about 0.1 to 5 mg / kg body weight.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • a composition is provided in a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • the antibody of the present invention may contain other drugs such as alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), antimetabolites (eg, methotrexate, 5_fluorouracil, etc.), anticancer antibiotics (eg, mitomycin, Adriamycin, etc.), plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etc.), cisplatin, carpoplatin, etopoxide, etc.
  • alkylating agents eg, cyclophosphamide, ifosfamide, etc.
  • antimetabolites eg, methotrexate, 5_fluorouracil, etc.
  • anticancer antibiotics eg, mitomycin, Adriamycin, etc.
  • plant-derived anticancer agents eg, vincristine, vindesine, taxol, etc.
  • cisplatin carpoplatin, etopoxide, etc.
  • the protein of the present invention can also be used as a cancer vaccine to activate the immune system of cancer patients.
  • a strong antigen-presenting cell eg, a dendritic cell
  • adoptive immunotherapy or the like in which the protein is phagocytosed and then returned to the patient's body after phagocytosis, is preferably applied.
  • the dendritic cells returned into the body can kill cancer cells by inducing and activating cytotoxic T cells specific for cancer cells.
  • the protein of the present invention may be, for example, a cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis) Safely administered to mammals (eg, humans, monkeys, mice, rats, rabbits, pigs) as vaccine preparations for the prevention or treatment of cancer, thyroid cancer, kidney cancer, brain tumors, blood tumors, etc. You can also.
  • a cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis
  • mammals eg, humans, monkeys, mice, rats, rabbits, pigs
  • vaccine preparations for the prevention or treatment of cancer, thyroid cancer, kidney cancer, brain tumors, blood tumors, etc. You can also
  • the vaccine formulation usually contains the protein of the invention and a physiologically acceptable carrier.
  • the carrier include liquid carriers such as water, saline (including physiological saline), buffers (eg, phosphate buffer), and alcohols (eg, ethanol). '
  • the vaccine preparation can be prepared according to a usual vaccine preparation manufacturing method.
  • the protein of the present invention is dissolved or suspended in a physiologically acceptable carrier.
  • the protein of the present invention and a physiologically acceptable carrier may be separately prepared, and these may be mixed and used at the time of use.
  • an adjuvant eg, aluminum hydroxide gel, serum albumin, etc.
  • a preservative eg, thimerosal, etc.
  • a soothing agent eg, glucose
  • cytotoxicity eg, inleucin-leukins such as interleukin-12, inleatherin-feron such as interferon-alpha, etc. is further added. You may mix.
  • the protein of the present invention When used as a vaccine preparation, the protein of the present invention may be used as an active form, but the protein of the present invention may be denatured to enhance antigenicity. Denaturation of the protein of the present invention is usually performed by heat treatment and treatment with a protein denaturant (eg, formalin, guanidine hydrochloride, urea). The resulting vaccine preparation has low toxicity, and may be usually administered as an injection, for example, subcutaneously, intradermally, or intramuscularly, or may be locally administered to or near a cancer cell mass.
  • a protein denaturant eg, formalin, guanidine hydrochloride, urea
  • the dosage of the protein of the present invention varies depending on, for example, the target disease, the subject of administration, the administration route, and the like.
  • the protein of the present invention is subcutaneously administered to an adult (body weight: 60 kg) suffering from cancer as an injection.
  • the dose is usually about 0.1 to 300 mg, preferably about 100 to 300 mg per dose.
  • the vaccine preparation may be administered once, but the vaccine preparation may be administered 2 to 4 times at intervals of about 2 weeks to about 6 months in order to increase the amount of antibody produced.
  • the present invention has a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • the exogenous DNA of the present invention or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the DNA transgenic animal of the present invention can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, the ribofection method It can be produced by transferring the desired DNA by a coagulation method, microinjection method, particle gun method, DEAE-dextran method or the like.
  • the exogenous DNA of the present invention is transferred to somatic cells, organs of living organisms, tissue cells, and the like by the DNA transfer method, and the cell culture and tissue culture are performed.
  • the DNA-transferred animal of the present invention can be produced by fusing these cells with the above-mentioned germ cells by a cell fusion method known per se.
  • Non-human mammals include, for example, red sea lions, bushes, higgins, goats, gray egrets, and birds. Nu, cats, guinea pigs, eight-star, mice, rats, etc. are used. Above all, rodents and biological cycles are relatively short in terms of the creation of disease animal model systems, and rodents that are easy to breed, especially mice (for example, pure strains such as C57BLZ6 and DBA2) As the crossing strain, a BeCSF strain, a BDFi strain, a BeDSFi strain, a BALB / c strain, an ICR strain, etc.) or a rat (eg, Wistar, SD, etc.) are preferable.
  • mice for example, pure strains such as C57BLZ6 and DBA2
  • a rat eg, Wistar, SD, etc.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
  • Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, addition or deletion of bases, DNA with substitution or the like is used, and also includes abnormal DNA.
  • the abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of a normal protein of the present invention is used.
  • the exogenous DNA of the present invention may be derived from a mammal of the same or different species as the animal of interest.
  • DNs derived from various mammals eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • having the DNA of the present invention having high homology to the DNA are transferred.
  • a DNA construct eg, a vector
  • a human DNA of the present invention is bound downstream of various promoters capable of expressing A into a fertilized egg of a target mammal, for example, a mouse fertilized egg
  • a DNA-transferred mammal that highly expresses the DNA of the present invention can be produced.
  • Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacterium such as ⁇ phage, a phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or Animal viruses such as baculovirus are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • promoters that regulate the expression of DN ⁇ include, for example, (i) viruses derived from viruses (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.).
  • viruses derived from viruses eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.
  • DNA promoters (ii) promoters derived from various mammals (human, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, perovrakin II, elastase, Erythropoietin, endoselin, muscle creatine kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor / 3, keratins Kl, K10 and K14, collagen types I and II, cyclic AMP Dependent protein kinase i3 I-subunit, dystrophy Insulin, tartrate-resistant alkaline rifos phosphatase, atrial natriuretic factor, endothelial receptor thymic synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATP) ase), nu
  • the above vector is a messenger R of interest in DNA-transferred mammals.
  • Yuichi Mineta a sequence that terminates the transcription of NA
  • a sequence of each DNA derived from a virus and various mammals can be used, and preferably The Simian virus SV40 Yuichi Mineta is used.
  • translation of splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc. to 5 'upstream of promoter region and promoter region for the purpose of further expressing the target foreign DNA Linking between regions or 3 ′ downstream of the translation region is also possible depending on the purpose.
  • the normal translation region of the protein of the present invention includes DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
  • Complementary protein prepared by known methods as all or part of genomic DNA from one of various commercially available genomic DNA libraries, or from RNA derived from kidney, kidney, thyroid cells, or fibroblasts
  • the foreign abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above-described cells or tissues by a point mutation induction method.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated to the downstream of the aforementioned promoter and optionally to the upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the transfer of the DNA indicates that all the progeny of the produced animal and the exogenous DN of the present invention will be present in all of the germinal cells and somatic cells.
  • the progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain exogenous DNA by mating, and should be subcultured as an animal having the DNA in a normal breeding environment. Can be done.
  • the transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germinal and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the DNA transfer indicates that all the offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of the germinal and somatic cells. Means that. The offspring of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germ cells and somatic cells.
  • the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately promoted by promoting the function of endogenous normal DNA. It may develop advanced disease and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, elucidation of the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and examination of methods for treating these diseases. It is possible.
  • a preventive / therapeutic agent for a disease associated with the protein of the present invention such as a cancer
  • a cancer Examples: colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor It can also be used for screening tests for preventive and therapeutic agents for blood tumors.
  • the non-human mammal having the foreign abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by the crossing. Can be done. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. D with promoter
  • the NA construct can be prepared by ordinary DNA engineering techniques. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • the offspring of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells.
  • the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately reduced by inhibiting the function of endogenous normal DNA.
  • Inactive refractory disease may occur, and the disease can be used as a si model animal. For example, it is possible to elucidate the pathological mechanism of the functionally inactive refractory state of the protein of the present invention and to examine a method for treating this disease using the abnormal DNA transgenic animal of the present invention.
  • the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention. Action).
  • a preventive / therapeutic agent for the protein of the present invention or a functionally inactive type refractory disease for example, Cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney It can also be used for screening tests of prophylactic and therapeutic agents for cancer, brain tumors, blood tumors, etc.).
  • Cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney It can also be used for screening tests of prophylactic and therapeutic agents for cancer, brain tumors, blood tumors, etc.).
  • cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture,
  • the DNA-transferred animal of the present invention in order to use the DNA-transferred animal of the present invention to develop a treatment for a disease associated with the protein of the present invention, including a refractory inactive type of the protein of the present invention, Using a test method and a quantitative method, it is possible to provide an effective and rapid screening method for a therapeutic agent for the disease. Further, using the DNA transfer product of the present invention or the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating a DNA associated with the protein of the present invention.
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • the DNA is inactivated by introducing a reporter gene (eg, a 3-galactosidase gene derived from Escherichia coli), and the reporter gene is controlled under the control of a promoter for the DNA of the present invention.
  • a reporter gene eg, a 3-galactosidase gene derived from Escherichia coli
  • a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, Alternatively, the DNA substantially does not have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention).
  • the knockout DNA of the present invention refers to embryonic stem cells of non-human mammals (hereinafter abbreviated as ES cells).
  • the method for artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. Due to these mutations, for example,
  • the knockout DNA of the present invention may be prepared by shifting the reading frame of the promoter or disrupting the function of the promoter or exon.
  • non-human mammalian embryonic stem cells of the present invention in which the DNA is inactivated include, for example, The DNA of the present invention possessed by the non-human mammal described above is isolated and its exon portion is a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or a lacZ galactosidase gene), cat (clo Inserting a reporter gene such as the ramphenicol acetyltransferase gene, etc., disrupts exon function, or terminates gene transcription in the intron between exons (eg, poly) A signal, etc.) to prevent synthesis of complete messenger RNA, A DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter, abbreviated as "gettering vector”) is introduced into the chromosome of the animal by, for example, homolog
  • the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like for example, those already established as described above may be used, and the method of the known Evans and Kaufma may be used. It may be newly established according to. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, it is an alternative pure immunological and genetic background.
  • BDF mice C57BLZ6 and DBA / 2 BDFi mice can be used favorably because they have a high number of eggs collected and their eggs are robust, and they have C57BLZ6 mice as their background.
  • the ES cells obtained as described above can be advantageously used when a pathological model mouse is created, because the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BLZ6 mice.
  • blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained.
  • male ES cells are generally more convenient for producing a germ line chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, than suffices ES cell number of about 1 colony (about 50), 'The primary selection of ES cells in the early stage of culture can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required in the early stage of culture.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative potential, but must be carefully subcultured because they tend to lose their ontogenetic potential.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5% oxygen
  • LIF 1-10000 U / ml
  • 5% CO 2, 90% air at about 37 ° C.
  • trypsin ZEDTA solution usually 0.01 to 0.5% trypsin Z 0.1 to 5 mM EDTA (Preferably about 0.1% Tribcine ZlmM EDTA) to obtain a single cell
  • a method of seeding on one cell is used.
  • Such subculture is usually performed every 1 to 3 'days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
  • ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
  • MJ Evans and MH Kaufman Nature, 292, 154, 1981; GR Martin, Proc. Natl. Acad. Sci. USA 78, 7634, 1981; Doetschman et al., Journal of Embryology 'and Experimental Morphology, Vol. 87, p. 27, 1985
  • cells of the present invention lacking the expression of the DNA of the present invention obtained by differentiating the ES cells of the present invention. Is useful in cell biology studies of the proteins of the invention in vitro.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal the same one as described above is used.
  • the non-human mammal deficient in expression of the DNA of the present invention can be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the evening-getting vector into the DNA of the present invention. Is knocked out by homologous recombination in which the DNA sequence in which is inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination be able to.
  • the cells in which the DNA of the present invention has been knocked out can be used as a target for the DNA sequence on the southern hybridization analysis or evening getter vector using the DNA sequence on or near the DNA of the present invention as a probe. It can be determined by analysis by PCR using the DNA derived from the mouse and the DNA sequence of the neighboring region other than the DNA of the present invention as a primer.
  • a non-human mammalian embryonic stem cell When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage. Hi And injected into the uterus of the pseudo-pregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
  • all tissues are more artificial than the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the DNA locus of the present invention in which a mutation has been added to, for example, by judging a color.
  • the individual obtained in this manner is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention. An individual with poor homo-expression can be obtained.
  • a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of an egg by a microinjection method. Compared to human mammals, it can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
  • the animal obtained by crossing is confirmed to be knocked out of the D and NA, and is bred in a normal breeding environment. Can do it.
  • the germline can be obtained and maintained according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained.
  • homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in DNA expression of the present invention Since it lacks various inducible biological activities, it can serve as a model for diseases caused by inactivation of the biological activity of the protein of the present invention, and is useful for investigating the causes of these diseases and studying therapeutic methods. It is.
  • the non-human mammal deficient in DNA expression of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • the present invention provides a method for screening a compound or a salt thereof, which has a therapeutic / preventive effect on diseases caused by the above, for example, cancer.
  • Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and the change in each organ, tissue, symptom of disease or the like of the animal is used as an index.
  • the therapeutic and prophylactic effects of the test compound can be tested.
  • test compound for example, oral administration, intravenous injection, or the like is used, and it can be appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal healing, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney Screening compounds with therapeutic and preventive effects against cancer, brain tumors, blood tumors, etc.
  • test compound When a test compound is administered to a non-human mammal deficient in the expression of DNA, the difference in the degree of onset of cancer and the degree of healing of cancer in the non-administered group of the test compound can be monitored over time in the above tissues. To observe.
  • test compound when administered to a test animal, the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
  • the test compound can be selected as a compound having a therapeutic and / or preventive effect on the above-mentioned diseases.
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic / therapeutic agent for the disease. Further, a compound derived from the compound obtained by the above-mentioned screening can be similarly used.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals). And the like, and especially preferred are physiologically acceptable acid addition salts.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, maleic acid) Acids, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, maleic acid
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
  • the preparations obtained in this way are safe and of low toxicity and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
  • the dosage of the compound or its salt varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound when the compound is orally administered, it is generally an adult (assuming a body weight of 60 kg).
  • the conjugate per day. l-100mg Preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered.
  • the single dose of the compound varies depending on the administration subject, target disease and the like.
  • the compound is usually in the form of an injection for an adult (with a body weight of 60 kg).
  • the dose can be administered in terms of weight per 60 kg.
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention and detecting the expression of a reporter gene.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention is inactivated by introducing a repo all-over-one gene.
  • a gene which can be expressed under the control of the promoter for the DNA of the present invention is used.
  • test compound examples include the same compounds as described above.
  • reporter gene the same one as described above is used, and a j8-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • the reporter By tracing the expression of a substance encoded by the gene, the activity of the promoter can be detected.
  • the tissue of the present invention expressing the protein of the present invention originally / 3-galactosidase is expressed instead of evening protein.
  • a reagent serving as a substrate for 3-galactosidase such as lyl / 3-galactopyranoside (X-gal)
  • the expression state of the protein of the present invention in an animal body can be easily determined. Can be observed.
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with daltaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or After reacting at 37 ° C for about 30 minutes to 1 hour, the ⁇ -galactosidase reaction is stopped by washing the tissue sample with ImM EDTA / PBS solution, and the color is observed. Just fine. Further, mRNA encoding 1 ac Z may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.
  • the compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, a).
  • a physiologically acceptable acid eg, an inorganic acid
  • a base eg, a
  • a salt with an alkali metal salt or the like is used, and a physiologically acceptable acid addition salt is particularly preferable.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid
  • benzoic acid methanesulfonic acid, benzenesulfonic acid, etc.
  • the compound of the present invention or a salt thereof that inhibits the promoter activity against DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein, and thus, for example, cancer (eg, colon cancer) , Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain, blood, etc.) It is useful as a preventive and therapeutic agent.
  • cancer eg, colon cancer
  • a compound derived from the compound obtained in the above-mentioned screening can also be used.
  • a drug containing the compound or a salt thereof obtained by the screening method is produced in the same manner as the above-described drug containing the protein of the present invention or a salt thereof. be able to.
  • the preparations obtained in this way are safe and of low toxicity and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult For a breast cancer patient weighing 60 kg, the compound is administered at about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg per day.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that inhibits promoter activity against DNA of the present invention is usually administered in the form of an injection.
  • the compound should be used in an amount of about 0.01 to
  • the dose can be administered in terms of weight per 60 kg.
  • the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of promoter of DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by DNA expression deficiency of the invention or the development of therapeutic agents.
  • transgene a DNA containing the entire promoter region of the protein of the present invention
  • genes encoding various proteins are ligated downstream thereof, and this is injected into an egg cell of an animal to produce a so-called transgene. If a nick animal (transgenic animal) is created, it will be possible to specifically synthesize the protein and examine its effects on the living body. Further, by binding an appropriate repo overnight gene to a portion of the above promoter and establishing a cell line that expresses the gene, the protein of the present invention itself can be specifically promoted or suppressed in its production ability in the body. It can be used as a search system for low-molecular compounds that have an action.
  • DNA Deoxylipo nucleic acid
  • RNA Liponucleic acid
  • a 1 a Alanine
  • Th r Threonine
  • HONB trihydroxy-5-norpolene-2,3-dicarpoxyimide DCC
  • SEMA4B indicates the nucleotide sequence of DNA including the full length gene encoding Ml. [SEQ ID NO: 7]
  • SEMA4B Shows the nucleotide sequence of DNA including the full length gene encoding M2. [SEQ ID NO: 1'0]
  • SEMA4B shows the amino acid sequence of M3.
  • Example 3 shows the base sequence of the antisense oligonucleotide used in Example 2, Example 3, Example 15 and Example 16.
  • Example 3 shows the nucleotide sequences of the oligonucleotides used in Example 2, Example 3, Example 15 and Example 16.
  • Example 3 shows the base sequence of the oligonucleotide used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • [SEQ ID NO: 21] 7 shows the nucleotide sequence of a primer used in Example 6.
  • Example 7 shows the amino acid sequence of peptide 1 used in Example 8.
  • Example 7 shows the amino acid sequence of peptide 2 used in Example 8.
  • Example 7 shows the amino acid sequence of peptide 3 used in Example 8.
  • Example 7 shows the amino acid sequence of peptide 4 used in Example 8.
  • the transformant Escherichia coli TOP10 / SEMA4B-Ml / pCR4-T0P0 obtained in Example 4 described below has been used since March 4, 2003 at 1-1, Tsukuba-Higashi 1-chome, Central No. 6 (mail number 305 -8566) at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, under the deposit number FERM BP-8316.
  • M2 / pCR4-T0P0 has been deposited at the Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology (AIST) at 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture since March 4, 2003. Deposited under number FERM BP-8317.
  • Example 1 The transformant Escherichia coli TOP 10 / SEMA4B-M3 / pCR4-T0P0 obtained in Example 4 described below has been used since March 4, 2003, at 1-1, Tsukuba-Higashi, Ibaraki Prefecture, Central No. 1 (postal number 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-8318.
  • AIST National Institute of Advanced Industrial Science and Technology
  • oligonucleotide microarray Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix
  • total RNA Table 1 extracted from 4 cases of cancer tissue and 5 cases of normal lung tissue.
  • the experimental method followed Affymetrix's experiment guide (Expression analysis technical manual).
  • Semaphorin 4B Semaphorin 4B
  • Semaphorin 4B-M1 Semaphorin 4B-M2
  • Semaphorin 4B-M3 Semaphorin 4B-M3
  • Lung cancer tissue (lot.0011-192-01293) 9.5 Lung cancer tissue (lot. 0011-192-01297) 1.
  • human non-small cell lung cancer cell line NCI-HI703 purchased from American Type Culture Collection (ATCC) was added to RPMI-1640 medium (containing 25 mM HEPES).
  • the antiserum in a 5% carbon dioxide gas stream, the antiserum is specifically expressed by the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 10
  • SEQ ID NO: 13 an antisense oligonucleotide sequence that hybridizes to the 3 ′ untranslated region sequence of the protein having the protein
  • anti-sense Abbreviated as sense oligonucleotide
  • control the reverse sequence (SEQ ID NO: 14) of the nucleotide sequence represented by SEQ ID NO: 13 was similarly phosphorothiated, purified by HPLC and used (hereinafter referred to as “control”). Abbreviated as oligonucleotide)).
  • the antisense oligonucleotide or control oligonucleotide diluted with Opti-MEM (Invitrogen) was diluted 5-fold with Opti-MEM (Invitrogen) and left at room temperature for 5 minutes. : 3 ratio
  • the 7th oligonucleotide (SEQ ID NO: 13) showed approximately 1.6 times the apoptosis-inducing activity as compared to the control oligonucleotide (SEQ ID NO: 14), showing a statistically significant difference. ⁇ 0.01) (Table 3).
  • SEMA4B antisense oligonucleotide reduces gene expression
  • the human non-small cell lung cancer cell line NCI-HI 703 used in Example 2 was suspended in the same medium as in Example 2, and a 24-well flat-bottom tissue culture was performed at a cell density of 60,000 cells / well (0.6 ml medium volume). The seeds were seeded on a culture plate (BD Falcon). After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, antisense oligonucleotides were transfected according to the method of Example 2.
  • the amount of oligonucleotide added was 240 L per well, and two types of antisense oligonucleotides (SEQ ID NO: 13 and SEQ ID NO: 15) and two types of control oligonucleotide (SEQ ID NO: 15) were used. No .: 14 and the oligonucleotides of SEQ ID NO: 16) were used.
  • antisense oligonucleotides and control oligonucleotides derived from SEQ ID NO: 15 and SEQ ID NO: 16 amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 10
  • SEQ ID NO: 15 amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 10
  • Reverse transcription was performed using Transcription Reagents (Applied Biosystems) according to the attached protocol.
  • a cDNA corresponding to 7 to 9 ng of total RNA was designated as type I, and SEMA4B and SEMA4B were prepared using two primers (SEQ ID NO: 17 and SEQ ID NO: 18) and SYBR Green PCR Master Mix (Applied Biosystems).
  • SEMA4B-M2 and SEMA4B-M3 genes was measured.
  • the expression amount of the 3-actin gene was measured using TaqMan i3-actin Control Reagents (Applied Biosystems) and used as an internal standard.
  • the non-transfection group When distilled water was used instead of the oligonucleotide solution (hereinafter referred to as the non-transfection group), the sum of SEMA4B, SEMA4B-MK, SEMA4B-M2 and SEMA4B-M3 gene expression was expressed as / 3-actin gene expression Of the antisense oligonucleotides (SEQ ID NO: 13 and SEQ ID NO: 15) were 0.98% and 1.1% in the administration group, which was statistically significant ⁇ 0.05). A decrease in the actual quantity was observed.
  • control oligonucleotide SEQ ID NO: 14 and SEQ ID NO: 16
  • the expression levels were 4.1% and 3.4%, and no statistically significant decrease in the expression level was observed as compared with the non-transfection group.
  • PCR was performed using two types of primers (SEQ ID NO: 19 and SEQ ID NO: 20).
  • the reaction solution 501 was composed of 11 of the above cDNA, 2.5 U PfuTurbo Hotstart DNA Polymerase (STRATAGENE), 1.0 M of each primer (SEQ ID NO: 19 and SEQ ID NO: 20), 200 M dNTPs, and 25 1 x GC Buffer I (Takara Shuzo).
  • the PCR reaction is repeated at 95 ° C for 1 minute, followed by 30 cycles of 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 4 minutes, followed by an extension reaction at 72 ° C for 5 minutes.
  • To add dATP to the 3 'end of the PCR reaction product 5 U of Ex Taq DNA Polymerase (Takara Shuzo) was added and incubated at 72 ° C for 7 minutes.
  • the obtained PCR reaction product was purified using a PCR Purification Kit (QIAGEN). This was subcloned into the plasmid vector pCR4-TOP0 (Invitrogen) according to the prescription of the T0P0 TA PCR Cloning Kit (Invitrogen).
  • SEQ ID NOS: 2, SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 11 correspond to nucleotides 1 to 237 and 2749 to 3766 of the SEMA4B gene (GeneBank Accession No. XM-0444533 gene).
  • the nucleotide sequences added to the 5, 5 and 3 'ends of the nucleotide sequence represented by, respectively, are shown as SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9 And SEQ ID NO: 12.
  • amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 2 completely matched the SEMA4B protein encoded by the SEMA4B gene (GeneBank Accession No. XM-0444533 gene).
  • SEMA4B-M3 is a protein containing SEMA4B-Ml; a protein containing the amino acid sequence (SEQ ID NO: 7) encoding the nucleotide sequence represented by SEQ ID NO: 8 is SEMA4B-M2; SEQ ID NO: 11 A protein having an amino acid sequence (SEQ ID NO: 1.0) encoding the base sequence represented by is designated as SEMA4B-M3.
  • the 90th g of the DNA sequence encoding SEMA4B corresponds to a, and the 111th g to a.
  • the 623th g is replaced by t, respectively, and the 623th substitution is accompanied by an amino acid substitution.
  • the 150th g of the nucleotide sequence of the DNA encoding SEMA4B corresponds to a, and the 489th g to a.
  • the 528th c is replaced with t
  • the 1266th is replaced with a
  • the 1588th c is replaced with a
  • the 2343th a is replaced with g
  • the 489th substitution is accompanied by an amino acid substitution.
  • nucleotide sequence of the DNA encoding SEMA4B-M3 (SEQ ID NO: 11)
  • the 1092th g of the nucleotide sequence of the DNA encoding SEMA4B (SEQ ID NO: 2) has been replaced with t, It is accompanied by amino acid substitution.
  • SEMA4B / pCR4-T0P0 a plasmid having a DNA having a nucleotide sequence represented by SEQ ID NO: 5 was converted to a DNA having a nucleotide sequence represented by SEMA4B-Ml / pCR4-T0P0, SEQ ID NO: 8
  • the plasmid having the DNA having the base sequence represented by SEMA4B-M2 / pCR4-TOP0 and SEQ ID NO: 11 was named SEMA4B-M3 / PCR4-TOP0, respectively.
  • the transformant into which the plasmid SEMA4B / pCR4-T0P0 was introduced was transformed into Escherichia coli TOP 10 / SEMA4B / pCR4-TOPO, and the transformant into which the plasmid SEMA4B-Ml / pCR4-TOPO was introduced was transformed into Escher i chi Transformants into which aco li TOP10 / SEMA4B-Ml / pCR4-TOPO and plasmid SEMA4B-M2 / pCR4-TOPO have been introduced are transformed into Escher i chia col i
  • Total RNA was prepared from the above 91 cell lines using RNeasy Mini Total Thigh Kit (QIAGEN). Using this 1 ⁇ tal RNA as a type II cDNA and preparing a cDNA by reverse transcription using random primers and performing a quantitative PCR reaction, the SEA4B gene (SEQ ID NO: 2) and the SEMA4B-M1 gene (SEQ ID NO: : Five ) ,
  • SEMA4B-M2 gene SEQ ID NO: 8
  • SEMA4B-M3 gene SEQ ID NO: 11
  • the PCR reaction was performed using the cDNA obtained from 3 to 4 ng of the above total RNA as type III, and the PCR reaction was performed under the same conditions as in Example 3, and SEMA4B, SEMA4B-MK, SEMA4B-M2 and SEMA4B-M3
  • SEMA4B, SEMA4B-MK, SEMA4B-M2 and SEMA4B-M3 The number of gene expression copies was calculated.
  • Table 4 shows the relative expression rates obtained by standardizing the expression level of the whole gene with the expression level of] 3-actin gene.
  • Plasmid SEMA4B / PCR4-TOP0 obtained in Example 4 was used as type III to amplify the PCR SEMA4B gene.
  • the composition of the reaction solution used was 2 ng of SEMA4B / pCR4-T0P0 as type II, 2.5 U of Pfu Turbo Hotstart DNA Polymerase (STRATAGENE), two types of primers (SEQ ID NO: 19 and SEQ ID NO: 2). 1) was added to each of 1 M, dNTPs was added to 200 ⁇ l, and lOx Pfu Buffer was added to 51 to make a liquid volume of 50 1.
  • PCR reaction a cycle of 95 ° C for 1 minute, 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 4 minutes was repeated 25 times.
  • the PCR reaction product was purified using a PCR Purification Kit (QIAGEN), and then treated with restriction enzymes Xbal and Eco RI. Plasmid p3xFLAG-CMV_14 (Sigma) was also treated with XbaI and EcoRI.
  • Each DNA fragment was purified using a PCR Purification Kit, and a ligation reaction was performed using a DNA Ligation Kit ver.2 (Takara Bio). After introducing the ligation reaction mixture into E. coli ⁇ 0 ⁇ 0, the transformed E.
  • An expression vector for animal cells expressing a protein in which a 3xFLAG tag was fused to the C-red end of the SEMA4B protein was constructed. Transformation was performed according to the method described in Example 6, except that the primer pair used for amplifying the SEMA4B gene by PCR was changed to another primer pair (SEQ ID NO: 19 and SEQ ID NO: 20). The selected E. coli was screened. As a result, a plasmid pCMV-14-SEMA4B-3xFLAG containing a cDNA fragment encoding a protein in which a 3xFLAG tag was fused to the C-terminal of the SEMA4B protein (SEQ ID NO: 1) was obtained.
  • SEQ ID NO: 1 Preparation and purification of peptide antibodies
  • SEMA4B protein SEQ ID NO: 1
  • SEMA4B-M1 protein SEQ ID NO: 4
  • SEMA4B-M2 protein SEQ ID NO: 7
  • SEMA4B-M3 protein SEQ ID NO: 10
  • amino acid sequence of peptide 1 [Asn-Ser-Ala-Arg-Glu-Arg-Lys-lie-Asn-Ser-Ser-Cys (SEQ ID NO: 22)] is the amino acid sequence of SEMA4B protein (SEQ ID NO: 1). This is a sequence in which Cys has been added to the C-terminus of the amino acid sequence from the 2nd to the 412th.
  • amino acid sequence of peptide 2 [Ser-Vat-Vat Ser-Pro-Ser-Phe-Vat-Pro-Thr-Gly-Glu-Lys-Pro-Cys (SEQ ID NO: 23)] is SEMA4B This is the sequence from the 582th position to the 596th position of the protein (SEQ ID NO: 1).
  • amino acid sequence of peptide 3 [Pro-Leu-Asp-His-Arg-Gly-Tyr-Gln-Ser-Leu-Ser-Asp-Ser-Pro-Cys (SEQ ID NO: 24)] is the SEMA4B protein (SEQ ID NO: 24). No .: This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from positions 781 to 794 in 1).
  • amino acid sequence of peptide 4 is the SEMA4B protein (SEQ ID NO: .. 1) This is a sequence in which Cys has been added to the C-terminal of the amino acid sequence from positions 797 to 809 of the protein.
  • the immunized animal used one male male egret KBL: JW (11-week-old, Oriental yeast), the first sensitization was complete Freund's adjuvant (Difco) suspension, and the second and subsequent times were incomplete Freund's adjuvant (Difco) The suspension was used. Sensitization was performed by subcutaneous injection into the back. One sensitization was performed using 0.5 mg of each antigen, and was repeated three times every 14 days after the first sensitization. On the 52nd day after the first sensitization, blood was collected from an artery under anesthesia to obtain about 50 ml of Suzuki.
  • the serum thus obtained was concentrated by ammonium sulfate salting out method,
  • the total amount of the obtained crude IgG fraction was purified by protein A affinity ram (Amersham-Bioscience), and about 103 mg and about 103 mg, respectively, were obtained from rabbits immunized with peptide 1, peptide 2, peptide 3 or peptide 4. 76 mg, about 112 mg and about 122 mg of purified IgG were obtained.
  • an IgG fraction that binds to a column on which each immunogenic peptide was immobilized was obtained.
  • Cys at the C-terminus of each peptide was used and coupled to a Sepharose column (Amersham-Bioscience) using a borate buffer.
  • the SEMA4B protein (SEQ ID NO: 1) was detected using the purified antibody prepared in Example 8 and a peptide antibody.
  • 1.5 ⁇ 10 6 NCI-H358 cells derived from human non-small cell lung cancer were suspended in 10 ml of RPMI-1640 medium (Invitrogen) containing 10% fetal calf serum (JRH) and seeded on a 10 cm diameter petri dish. The cells were cultured at 37 ° C under a 5% carbon dioxide gas flow.
  • Phosphatase Inhibitor Cocktail-2 (Sigma)] was added and left at 4C for 30 minutes. Collect the RIPA buffer and centrifuge at 15,000 rpm for 20 minutes. The liquid was used as a cell-free extract.
  • This cell-free extract and sample buffer for double concentration SDS-PAGE (125 mM Tris' hydrochloric acid buffer, ⁇ 6.8, 40% glycerol, 4% SDS, 0.04% bromophenol blue and 5% 2-mercapto [Ethanol] was mixed in equal volumes and heated at 95 ° C for 5 minutes, and then 10x1 was subjected to SDS-PAGE on a 10% acrylamide gel.
  • the proteins separated by electrophoresis were transferred to CLEARBUT® membrane (ATT0) according to a conventional method, and then a blocking solution [50 mM Tris. In a hydrochloric acid buffer, pH 7.5, 500 mM sodium chloride, 0.1% Tween 20, 5% skim milk] for 1 hour at room temperature.
  • a blocking solution [50 mM Tris. In a hydrochloric acid buffer, pH 7.5, 500 mM sodium chloride, 0.1% Tween 20, 5% skim milk] for 1 hour at room temperature.
  • the peptide antibody AS-2531, AS-2532, AS-2591 or AS-2592 prepared in Example 8 was diluted with a blocking solution so as to have a concentration of 3 g / ml. Reacted. Subsequently, it was left at room temperature for 1 hour in an HRP-labeled anti-Egret IgG antibody (Amersham-Bioscience) diluted 50,000 or 100,000 with a blocking solution.
  • SEMA4B protein was detected
  • Example 7 Using the plasmid pCMV-14-SEMA4B-3xFLAG obtained in Example 7, a cell-free extract was prepared in the same manner as in Example 9. A cell-free extract 400 was added to a suspension 501 in which Protein G-Sepharose FF (Amersham-Bioscience) was suspended in an equal volume of RIPA buffer, and the peptide antibodies AS-2531, AS -2532,
  • a mixed solution to which either one of AS-2591 or AS-2592 was added was prepared, and stirred at 4 ° C. After washing the protein G-Sepharose 4FF co-precipitated fraction with RIPA buffer, 501 sample buffer for SDS-PAGE (62.5 mM Tris-HCl buffer, pH 6.8, 20% glycerol, 2% SDS , 0.02% bromophenol alcohol and 2.5% 2-mercaptoethanol] and heated at 95 ° C. for 5 minutes, and then 51 or 10 I was subjected to SDS-PAGE on a 10% acrylamide gel. Detection was in accordance with the method described in Example 9.
  • a mouse anti-FLAG M2 antibody (Sigma) diluted to 0.2 g / ml or 0.1 g / ml with a blocking solution was used as the primary antibody, and an HRP-labeled anti-mouse IgG antibody (Amersham-Bioscience) was used as the primary antibody. Those diluted 15,000-fold or 50,000-fold with a blocking solution were used as secondary antibodies.
  • immunoprecipitation was performed using any of the peptide antibodies AS-2531, AS-2532, AS-2591 and AS-2592, a specific band derived from the SEMA4B protein was observed near the molecular weight of 100kD.
  • H1703; ovarian cancer cell lines SK0V-3 and TOV-21G; prostate cancer cell line DU145; and JI-extracted cancer cell line PANC-1 were cultured on two 10 cm diameter Petri dishes.
  • One petri dish of each cell was dispersed with trypsin-EDTA (Invitrogen), and the number of cells was counted. Based on the number of cells counted, add 1 ml of ice-cold RIPA buffer (described in Example 9) to 5 ⁇ 10 6 cells to one remaining Petri dish, and add 4 ° C For 30 minutes. The RIPA buffer was recovered, and the supernatant obtained by centrifugation at 15, OOikpm for 20 minutes was used as a cell-free extract.
  • the Size TM X ProteinG Immunoprecipitation Kit Pieris
  • Cells were harvested from the wells in which one to three cells had proliferated and formed colonies, and were seeded equally in 2 wells of a 48 well plate. After culturing was continued until the cell density reached 50% or more, 50 ⁇ l of the sample buffer for SDS-PAGE described in Example 10 was added to 1 ⁇ l of cells to prepare a cell lysate. After heat treatment at 95 ° C for 5 minutes, 5x1 was subjected to SDS-PAGE on a 10% acrylamide gel. According to the method described in Example 9, Western blotting was performed using the peptide antibody AS-2532 to search for stably expressing cells that constitutively express the SEMA4B protein (SEQ ID NO: 1).
  • the cells recovered from the other well were diluted to 0.7 cells per well, and then seeded on a 96-well plate.
  • the culture was continued at 37 ° C. under a 5% carbon dioxide gas flow while changing the G418 selection medium every 3 or 4 days until the cell density reached about 50%.
  • inoculate the same amount in a 1-well plate of 48-well plate, continue culturing until the cell density becomes 50% or more, and use the cell lysate prepared from 1-well cells in the same manner as described above. Blotting was performed.
  • a clone that highly expressed the SEMA4B protein (SEQ ID NO: 1) was selected to obtain a SEMA4B / S358 stable cell line.
  • Biotin was labeled using Immunoprecipitation Kit (Roche Diagnostics). Subsequently, using 1 ml of the cell-free extract prepared according to the method of Example 9 and 5 g of the peptide AS-2591 prepared in Example 8, immunoprecipitation was performed according to the method of Example 10, followed by SDS-PAGE. Was done. Detection using HRP-labeled streptavidin (Amersham-Bioscience) revealed a band derived from the SEMA4B protein near the molecular weight of lOOkD. The SEMA4B protein, SEMA4B-Ml protein, and SEMA4B-M2 protein And that the SEMA4B-M3 protein was localized on the cell surface.
  • the human non-small cell lung cancer cell lines NCI-H2228 and NCI-H358 and SEMA4B / H358 described in Example 12 were each seeded on a Petri dish having a diameter of 10 cm, and cultured until they became subconfluent. After washing each cell with PBS, PBS containing 0.5% BSA and 5 mM EDTA was added, and the mixture was left at room temperature for 15 minutes to disperse the cells. Next, use Buffer A (HBSS containing 2% fetal bovine serum (JRH) and 0.1% sodium azide (Hanks' Balanced Salt Solutions, Invitrogen)) at 4 ⁇ 10 6 cells / ml.
  • Buffer A HBSS containing 2% fetal bovine serum (JRH) and 0.1% sodium azide (Hanks' Balanced Salt Solutions, Invitrogen)
  • the cells were suspended to a final concentration of 10 mg / ml, and AS-2532 or non-immune rabbit (Jackson) was added to a final concentration of 10 g / ml, and left on ice for 3 hours. After washing the cells with buffer A, the cells were suspended in buffer A containing 10 g / ml of Alexa488-labeled anti-Peacock IgG pups (Molecular Probes), and left on ice for 2 hours. After washing again with buffer A, analysis was performed with FACScan (BD Biosciences).
  • NCI-H358 was suspended in RPMI-1640 medium (Invitrogen) containing 10% fetal calf serum (JRHRH, ImM sodium pyruvate and 25 mM HEPES), and the cell density was 8 ⁇ 10 3 cells / ⁇ (medium volume) 80 / ⁇ 1) NCI-H358 was seeded on a 96-well flat bottom tissue culture plate (BD Falcon) and cultured overnight at 37 ° C in a 5% charcoal gas stream.
  • RPMI-1640 medium Invitrogen
  • JRHRH 10% fetal calf serum
  • HEPES fetal calf serum
  • the apoptosis-inducing activity of the above oligonucleotide was measured according to the attached protocol of Diagnostics) and Caspase-Glo 3/7 assay (Promega II).
  • the antisense oligonucleotide showed 1.42 and 1.77 times the apoptosis-inducing activity, respectively, compared to the control oligonucleotide used as a negative control, with statistically significant differences (P ⁇ 0.01) (Tables 5 and 6).
  • Apo Bok one cis-inducing activity (A 4 5 -. A 492 ) Mean value Standard deviation blank 0.2 1 7 0.007 Con Bok roll oligonucleotides 0.330 0.041
  • RPMI-1640 medium (Invitrogen) containing 10% fetal calf serum (JRH), ImM sodium pyruvate and 25 mM HEPES was used.
  • ACL-4 medium (ATCC) containing 10% FBS was used for CI-H1651.
  • NCI-H23 contains RPMI-1640 medium containing 10% fetal bovine serum (JRH) and 25 mM HEPES. (Invitrogen) was used.
  • LipofectAMINE transfection reagent (Invitrogen) 0.41 was added, and the mixture was further left at room temperature for 15 minutes. The whole amount of the mixture was added to the culture solution of each cell, and the culture was continued for 3 days, and then the apoptosis-inducing activity of the above oligonucleotide was measured according to the protocol attached to Cell Death Detection ELISA PUiS (Roche Diagnostics).
  • the antisense oligonucleotide was compared to the control oligonucleotide used as a negative control, respectively.
  • NCI-H2228) shows 1.58-fold (NCI-H2228), 1.21-fold (NCI-H1651) and 1.25-fold (NCI-H23) apo1 cis-inducing activity, with a risk factor of P ⁇ 0.05 (NCI-H228), P ⁇ 0.05 (NCI-H228). -H1651) and P ⁇ 0.01 (NCI-H23).
  • the results showed statistically significant differences (Table 7, Table 8, and Table 9).
  • Cis-inducing activity (A 4 Q 5 — A 492 ) Mean standard deviation Blank 0.5 2 3 0. 0 9 1 Control oligonucleotide 1. 1 5 2 0. 1 0 1
  • Apoptosis inducing activity (A 4 Q 5 — A 492 ) Mean Standard deviation Blank 0.6 0.78 0.02 8 Control oligonucleotide 1.0 8 1 0. 0 5 0
  • Antisense Sense Rigonucleide 1.3 5 1 0. 0 5 8
  • the human non-small cell lung cancer cell line NCI-H2228 was treated with the egret peptide antibodies AS-2531 and AS-2532 obtained in Example 8, and the apoptosis-inducing activity of these egret peptide antibodies was measured.
  • NCI-H2228 was suspended in RPMI-1640 medium (Invitrogen) containing 10% fetal calf serum (JRH), ImM sodium pyruvate and 25 mM HEPES to a cell density of 4 ⁇ 10 3 cells / well.
  • the cells were seeded on a 96-well flat bottom tissue culture plate (BD Falcon) coated with type I collagen, and cultured at 37 in a 5% carbon dioxide gas stream.
  • the egret peptide antibodies (AS-2531, AS-2532, and non-immune egret IgG Oackson) obtained in Example 8 were diluted with PBS, and the final concentration of each antibody was 15 g / ml, 458/1111 and 150 ⁇ g / ml.
  • the protein used in the present invention is specifically expressed in cancer cells, and is a diagnostic marker for cancer. Therefore, a compound or a salt thereof that inhibits the activity of the protein, a compound or a salt thereof that inhibits the expression of the protein gene, an antisense polynucleotide of the present invention, and an antibody of the present invention include, for example, cancer (eg, colon cancer, Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain, blood, etc. ) Prevention-Can be used safely as a therapeutic agent, apoptosis promoting (inducing) agent, etc.
  • cancer eg, colon cancer, Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain, blood, etc.
  • Prevention-Can be used safely as a therapeutic agent
  • the protein used in the present invention or the polynucleotide encoding the same, the antibody of the present invention, etc. may be used for cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen)
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen
  • cancer eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen
  • prophylactic and therapeutic agents for cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, blood tumor,

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Abstract

La présente invention concerne un composé qui inhibe l'expression d'une protéine qui a une séquence d'acides aminés qui est identique ou sensiblement identique à la séquence d'acides aminés représentée par SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 ou SEQ ID NO:10, ou l'expression d'un gène de la protéine, un polynucléotide anti-sens ayant une séquence de bases qui est complémentaire ou sensiblement complémentaire à la séquence de bases d'ADN qui code pour la protéine mentionnée ci-dessus ou son fragment peptidique ou une partie de la séquence de bases mentionnée ci-dessus, un anticorps dirigé contre la protéine mentionnée ci-dessus ou son fragment peptidique, etc., qui sont utiles en tant qu'agents de prévention ou de traitement du cancer, etc., en tant qu'agents pour promouvoir l'apoptose etc.
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WO2005118631A1 (fr) 2004-06-03 2005-12-15 Takeda Pharmaceutical Company Limited Nouveau complexe protéinique et utilisation de ce complexe
WO2006014999A2 (fr) * 2004-07-27 2006-02-09 Five Prime Therapeutics, Inc. Compositions et methodes d'utilisation de modulateurs de nectine 4, de semaphorine 4b, d'igsf9 et de kiaa0152 dans le traitement de maladies

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WO2000012708A2 (fr) * 1998-09-01 2000-03-09 Genentech, Inc. Nouveaux pro-polypeptides et sequences correspondantes
WO2000078961A1 (fr) * 1999-06-23 2000-12-28 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant pour ces polypeptides
WO2001036440A1 (fr) * 1999-11-19 2001-05-25 Human Genome Sciences, Inc. 23 proteines humaines secretees
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WO2001077137A1 (fr) * 2000-04-12 2001-10-18 Human Genome Sciences, Inc. Proteines de fusion d'albumine
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WO2005061704A1 (fr) * 2003-12-24 2005-07-07 Takeda Pharmaceutical Company Limited Substance destinee a la prevention et au traitement du cancer
WO2005118631A1 (fr) 2004-06-03 2005-12-15 Takeda Pharmaceutical Company Limited Nouveau complexe protéinique et utilisation de ce complexe
WO2006014999A2 (fr) * 2004-07-27 2006-02-09 Five Prime Therapeutics, Inc. Compositions et methodes d'utilisation de modulateurs de nectine 4, de semaphorine 4b, d'igsf9 et de kiaa0152 dans le traitement de maladies
WO2006014999A3 (fr) * 2004-07-27 2006-12-21 Five Prime Therapeutics Inc Compositions et methodes d'utilisation de modulateurs de nectine 4, de semaphorine 4b, d'igsf9 et de kiaa0152 dans le traitement de maladies

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